SELEX (Systematic Evolution of Ligands by EXponential enrichment) is an in vitro selection method used to generate high affinity ligands for specific target compounds (Ellington A D, Szostak J W (1990) Invitro Selection of Rna Molecules That Bind Specific Ligands. Nature 346 (6287):818-822; Joyce G F (1989) Amplification, mutation and selection of catalytic RNA. Gene 82 (1):83-87; Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249 (4968):505-510). These selected molecules, called aptamers, are derived from large libraries of nucleic acids with random sequences through an iterative process of binding, partitioning, and amplification of sequences that bind to the target. This process enriches the initial random library for higher binding affinity sequences, and the cycle is repeated until the molecules in the enriched pools converge on the highest affinity sequence. Since this method was first introduced, aptamers have become valuable tools in biotechnology, diagnostics and therapeutics (Tombelli S, Mascini M (2009) Aptamers as molecular tools for bioanalytical methods. Current opinion in molecular therapeutics 11 (2):179-188).
There is interest in improving SELEX technology to obtain highly specific aptamers much more rapidly. However, despite their potential, many technologies are difficult to scale for multiplexed or parallel selections. For example, Park et al. and Ahn et al. used microfluidic sol-gel devices that could utilize up to five targets for multiplexing (Park S M, Ahn J Y, Jo M, Lee D K, Lis J T, Craighead H G, Kim S (2009) Selection and elution of aptamers using nanoporous sol-gel arrays with integrated microheaters. Lab on a chip 9 (9):1206-1212; Ahn J Y, Jo M, Dua P, Lee D K, Kim S (2011) A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection. Oligonucleotides 21 (2):93-100), but currently no large-scale microfluidic selections have been demonstrated. Large-scale parallel selections have been done with microplate technologies, which are of particular interest due to the availability of protocols and automated liquid handling devices (Cox J C, Ellington A D (2001) Automated selection of anti-protein aptamers. Bioorganic & medicinal chemistry 9 (10):2525-2531; Cox J C, Rudolph P, Ellington A D (1998) Automated RNA selection. Biotechnology progress 14 (6):845-850). However, in contrast to microfluidic devices that utilize flow and other dynamic behavior, most of these selections rely on traditional equilibrium solution binding (Eulberg D, Buchner K, Maasch C, Klussmann S (2005) Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist. Nucleic Acids Res 33 (4):e45) or interactions with target molecules that are bound or adsorbed to the plate surface (Drolet D W, Jenison R D, Smith D E, Pratt D, Hicke B J (1999) A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX). Combinatorial chemistry & high throughput screening 2 (5):271-278; Jolma A, Kivioja T, Toivonen J, Cheng L, Wei G, Enge M, Taipale M, Vaquerizas J M, Yan J, Sillanpaa M J, Bonke M, Palin K, Talukder S, Hughes T R, Luscombe N M, Ukkonen E, Taipale J (2010) Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome research 20 (6):861-873).
Despite advances toward more sophisticated and automated SELEX, little has been done to characterize and optimize new or current technologies, and recent binding studies show significant discrepancies with existing theory (Daniel C, Roupioz Y, Gasparutto D, Livache T, Buhot A (2013) Solution-Phase vs Surface-Phase Aptamer-Protein Affinity from a Label-Free Kinetic Biosensor. PloS one 8 (9):e75419; Latulippe D R, Szeto K, Ozer A, Duarte F M, Kelly C V, Pagano J M, White B S, Shalloway D, Lis J T, Craighead H G (2013) Multiplexed microcolumn-based process for efficient selection of RNA aptamers. Analytical chemistry 85 (6):3417-3424). Therefore, empirical methods have been used to optimize selection conditions and aid the development of new models (Ozer A, White B S, Lis J T, Shalloway D (2013) Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection. Nucleic Acids Res 41 (14):7167-7175). As new high-throughput technologies emerge, these studies will become even more important in order to obtain the most effective and robust selections under the available parameters.
The present invention is directed to overcoming these and other deficiencies in the art.